A.

General Precautions to be taken in the laboratory

1. Care should be taken to ensure that all gas taps work efficiently and that no gas
escapes when the taps are turned off. Do not attempt to find gas leaks by using
a lighted match stick.
2. Electric switches and connections should be repeatedly inspected and kept free
from corrosion, so that no danger shall arise from short circuiting or due to the
exposure of naked wire from which the insulation has been broken or corroded
away.
3. When a laboratory is to be left unattended for any length of time all gas and
water taps should be turned off.
4. Paper should never be used to carry light from one burner to another. Insoluble
waste should not be thrown into the sink.
5. If corrosive or poisonous liquids are being thrown down into the sink they
should be accompanied by a generous supply of water to ensure that by the time
they reach the main drain they will be too diluted to be dangerous.
6. When working in a laboratory it is better to wear some sort of protective coat,
apron or overcoat.
7. All bottles and containers should be carefully and distinctly labelled so that no
confusion or possible error can arise when several containers are being filled
with different chemicals.
8. Bottles should never be carried by the neck. Trays or polythene buckets or
suitable trolley should be used.
9. When dealing with volatile inflammable liquids no flame should be allowed in
the vicinity and care should be taken to avoid inhalation of vapour.
10. It is often an advantage to use glass rod to direct the liquid stream when pouring
from one bottle to another.
11. When diluting concentrated sulphuric acid always add the acid to the water kept
in ice and not vice versa. Add the acid slowly, preferably down a glass rod with
stirring to reduce the violence of the reaction.
12. When NaOH is dissolved in water heat is evolved. Use cold water and add
NaOH to it little by little. Do not handle NaOH with unprotected hands.
 2

13. Ether or any other volatile inflammable solvents should not be heated directly
over a nacked flame. Use water baths or heating mantles.
14. When pushing a glass tube or rod through a hole in rubber cork lubricate well
with water.
15. Poisonous, radio active and offensive reagents should never be pipetted by
mouth. Use propipette.
16. It is not advisable to use glass stoppers in bottles containing KOH and NaOH.
The stoppers are apt to stick and their removal involve breaking the neck of the
bottle. Use rubber stopper or store item in polythene containers.
17. Don’t use volumetric flasks for heating or boiling the solutions.
18. Use the glass bids to avoid bumping.
 3

B. Food Analysis

Foods are the substances which, when consumed by humans, are utilized
by the body in its normal metabolic activities. Foods influence growth, nutrition,
well being and even reproduction. They may also increase or decrease
susceptibility to certain infections.
Foods contain various nutrients- proteins, carbohydrates, fats, minerals and
vitamins- which contribute in different ways to body need. In addition, they
contain indigestible materials which aid in peristalsis; water which serves as a
vehicle for transporting food and waste products, assists in the regulation of the
body temperature and takes part in many chemical processes. Most foods contain
almost all the nutrients in various proportions, some foods being rich in certain
nutrients. Foods contain some toxic substances also which, if present, inhibit the
normal metabolic processes of other essential nutrients in the body. As well food
products are developed or prepared after treating the food materials by different
processing methods like soaking, cooking, decortication, germination, fermentation
etc. which affect the nutrient content of the products. Hence, it is very essential to
analyse the food and food products to know the actual nutrient content and effect
of different processing methods on the nutrient and toxic substances of food so that
right kind of food after the application of proper processing techniques can be
consumed.
Food analysis deals with the principles, methods and techniques necessary
for quantitative physical and chemical analysis of the food products and
ingredients.

C. Sampling procedure

The perfect sample is 100 per cent of the material being analyzed. But it is
practical only when the quality of material for which an analysis is desired is small
enough to be used as a laboratory sample. Usually it is necessary to abstract from a
quantity of material, a laboratory sample should be the representative of entire
 4

material. If the entire material is homogenous, it is not difficult to secure a

representative sample.
But in case of heterogeneous material, while securing the representative
sample, one should take care of the quantity of material in the lot, type of container
or containers, and conditions under which the material has been stored.

1. Number of portions for a composite sample -

When an analysis is desired, samples are abstracted from different portions
of material or from containers of the entire lot of material. The several individual
samples are combined, thoroughly minced, and a laboratory sample is abstracted
which will be the representative of the composite of samples. To facilitate mixing
the composite when it consists of dry, granular materials of widely different particle
size, it may be necessary to grind the composite sample before mixing.

2. Samplers
Several instruments or samplers are used for the proper sampling of various
materials. Of these, the most important are -
a) Thief - Used in sampling liquids.
b) Trier - Used to obtain cross-section sample
c) Probe from dry or plastic material.
d) Sampling tube
e) Auger or drill - Used to remove samples from solid
material.
f) Pelican - Used to abstract samples from a
continuous flow of material.

3. Reducing the composite sample to laboratory size

The reduction of the composite sample to laboratory size is accomplished by
passing the material through a divider or riffle. In case of unavailability of such
apparatus, the same result may be attained through mixing and quartering by hand,
mixing of dry, finely divided, granular material is accomplished by placing the
composite sample in the centre of large sheet of paper or cloth. The sample is
thoroughly mixed.
 5

When the material has been mixed sufficiently, the pile is made
approximately circular and divided into quarters. Two opposite quarters are saved
and the other two are discarded. The two quarter saved are again mixed and
quartered and the process repeated, until a quantity of material is obtained of
laboratory sample size.

4. Grinding to correct particle size

For the material that cannot be properly mixed and quartered, it must first be
ground to a state of fineness in order to be mixed thoroughly. There are various
types of grinding apparatus for the purposes - Willey mill, mortar and pestle,
mechanical high-speed beaters or blender and meat grinders.
Pulpy material such as fruit and vegetables, frozen desserts containing nuts
and fruits, and meat, may be disintegrated and converted into a homogenous sample
by means of mechanical beaters or mixers.

5. Mixing & sampling liquids

Liquids may be mixed easily by stirring, shaking or pouring from one
container to another. If sample are remained immediately after cessation of mixing,
they will be representative of the whole.
 6

D. Proximate analysis of food

A proximate analysis of a food or food product involves the determination

of the percentage of moisture, ash, crude fat, crude fibre, crude protein, and
carbohydrate content in the food.

1. Determination of moisture content

Temperature and pressure govern the amount of moisture obtained by drying

methods. As a result, drying method involves the use of heat alone, heat and
reduced pressure, or drying agents and reduced pressure.
Materials required
Desiccator, weighing bottle, drying oven, weighing balance.
Procedure
Dry the weighting bottle, cool in a desiccator and weigh. Transfer
approximately 2 to 10 g of the sample to the bottle, cover and weigh. Remove the
cover and place it loosely on the dish. Dry it in an oven at 100 to 105oC, cool in a
desiccator and weigh. Continue the process till the constant weight is obtained.

Calculations
Weight of the bottle = W1
Weight of the bottle + food sample = W2
Weight of the bottle + food sample (after attaining = W3
concurrent weight)
Weight of sample = W2 - W1
Weight of moisture = W2 – W3

W2 – W3
Moisture % = x 100
W2 - W1
 7

1. Determination of ash content

Mineral elements in food and food products are found in organic and
inorganic combinations. Inorganic salts such as phosphates, carbonates, chlorides,
sulphates, and nitrates of sodium, potassium and calcium are common. Salts of
organic acids, such as malic, oxalic, acetic, pectic etc. may also be present. In
addition, certain of the mineral elements may be built into complex organic
molecules.
Materials required
Crucible, clay pipe, muffle furnace, desiccator, weighing balance, asbestos
sheet, spatula, tong.
Procedure
Weigh 5 to 10 g sample in a previously ignited and weighed crucible.
Carefully ignite the sample over a burner and heat until the sample is thoroughly
charred. Transfer the crucible and contents to a muffle furnace held at a dull red
heat (550o – 600oC) and continue the ashing process until the ash has a grey - white
appearance. Cool in a desiccator and weigh. Repeat the process at an interval of
one hour until a constant weight is obtained.

Calculation
Weight of crucible = W1
Weight of crucible + sample = W2
Weight of crucible + sample = W3
(after removing from muffle furnace)
Weight of sample = W2 - W1
Weight of ash = W3 – W1

W3 – W1
Ash % = x 100
W2 - W1
 8

1. Determination of crude fat content

There are various methods available to determine the fat and all the fat
soluble materials present in the sample. These fat soluble materials may include
phospholipids, sterols, free fatty acids, carotenoid pigments, chlorophyll etc. in
addition to fat. It is why the determination is frequently referred as the crude fat.
The material to be analysed must be dried thoroughly in an oven and the solvent
used for the extraction must be anhydrous. This is necessary to avoid the presence
of any water so that water soluble materials are not extracted and determined with
the fat. Hence, the fat determination is made to follow a moisture determination and
the dried sample is used directly for the determination of fat.
Materials required
Soxhlet apparatus, heating mantles, thimbles, fat free cotton, weighing
balance & oven.
Reagents
Mixture of Petroleum ether and diethyl ether (1:1)
Procedure
Remove a dried 250 ml soxhlet flask from the oven and after cooling in a
desiccator weigh it. Repeat the procedure until consecutive concurrent values are
obtained. Weigh 5 g of the moisture free powdery sample directly into a filter
extraction thimble and plug the end of the thimble with fat free cotton. Place the
thimble and contents in the central syphon portion of the soxhlet apparatus. Put 160
ml of analytical grade petroleum ether and diethyl ether mixture (1:1) into the flask
and connect it to the soxhlet syphon and condenser. Reflux for five hours at 60oC.
Allow the thimble and contents to soak in ether for 24 hours. Plug the condenser
with moistened cotton. Distill off the ether and place the flask and contents in an
oven at 105oC. Dry for three hours, cool the flask & contents in a desiccator and
when it gets cool weigh it. The fat content can be calculated from the weight of
material held in the receiver flask.
Calculations
Wt of the empty soxhlet flask = W1
Wt of the empty flask with fat = W2
Wt of fat = (W2 - W1)
W2 - W1
Fat content (%) = x 100
Wt of sample
 9

4. Determination of crude fiber content

The crude fiber content is an index of the amount of indigestible matter, or
roughage, in a food stuff. It is made up of cellulose, lignin, pentosans and a little
nitrogenous matter. Although fiber has no appreciable food value, it functions in
the intestinal tract to give bulk to the contents and so stimulates intestinal peristaltic
action.
Principle: The crude fiber is estimated by first digesting the sample with
boiling dilute acid to hydrolyse the carbohydrate and protein materials contained in
it. Further digestion with boiling dilute alkali causes the saponification of the fatty
materials not extracted by ether.
Materials required : Beaker, Glass rod, muslin cloth, crucible, muffle furnace
Reagents
1. 0.225 N sulphuric acid(1.25%)
2. 0.313 Sodium Hydroxide(1.25%)
Procedure
About 2-5 g of moisture and fat free sample are weighed into a 500 ml
beaker and 200 ml of boiling 0.255 N (1.25% W/V) sulphuric acid is added. The
mixture is boiled for 30 min. keeping the volume constant by the addition of water
at frequent intervals (a glass rod is inserted in the beaker to help smooth boiling).
At the end of this period, the mixture is filtered through a muslin cloth and the
residue is washed with hot water till it becomes free from acid. The material is then
transferred to the same beaker and 200 ml of boiling 0.313 N (1.25%) NaOH is
added. After boiling for 30 min (keeping the volume constant as before) the
mixture is filtered through muslin cloth. The residue is washed with hot water till it
becomes free from alkali, followed by washing with some alcohol and ether. It is
then transferred to a crucible, dried overnight at 80-100oC and weighed (We). The
crucible is heated in a muffle furnace at 600oC for 2 to 3 hr, cooled and weighed
again (Wa). The difference in the weights (We-Wa) represents the weight of crude
fibre.
Calculation
100 - (moisture* + fat*) x Wt of fibre
Crude fibre (g/100 g sample) =
Wt of sample taken (moisture and fat free)

*g/100 g sample.
 10

5. Determination of crude protein content

Macro-Kjeldahl method

Principle
The estimation of nitrogen is done by Kjeldahl method which depends upon
the fact that organic nitrogen when digested with sulphuric acid in the presence of a
catalyst (selenium oxide, mercury or copper sulphate) is converted into ammonium
sulphate. Ammonia liberated by making the solution alkaline is distilled into a
known volume of a standard acid, which is then back-titrated. The protein content
is obtained by multiplying the nitrogen value with 6.25.
Reagents
1. Digestion mixture : 98 parts K2SO4 + 2 parts CuSO4
2. 40% NaOH
3. N/10 NaOH
4. N/10 H2SO4
5. Methyl red indicator : 0.1 g of the indicator dissolved in 60 ml of alcohol and
water added to make to 100 ml.
Procedure
The sample (0.5 - 2.0 g) is weighed into a dry Kjeldahl flask. About 5 g of
digestion mixture and 20 ml of pure conc. H2SO4 are added to the same sample and
the mixture is digested by heating for 4 to 5 hr. Glass beads are added to prevent
bumping. After the contents of the flask become clear, the digestion is continued
for at least 1 hr. The contents of the Kjeldahl flask are cooled, diluted with distilled
water and the mixture is made alkaline by adding excess of 40% NaOH (about 75
ml). A small quantity of pumice powder is added to prevent bumping during
distillation. The ammonia liberated is distilled into a receiver containing 25 ml of
N/10 H2SO4. The excess of acid in the receiver is back-titrated against N/10 NaOH
using 3 drops of methyl red indicator.
A reagent blank is similarly digested and distilled. The titre value is
subtracted from the value obtained for the sample to get the true titre value ‘b’.
 11

Calculation
If ‘a’ g of the sample are taken and if ‘b’ and ‘c’ ml of alkali of normality ‘d’
are required for back-titration and to neutralise 25 ml of N/10 H2SO4 respectively,
then the

(c-b) x 14 d x 6.25
Protein content in g/100 g sample is x 100
a x 1000

6. Determination of carbohydrate content

The value obtained by subtracting the sum of the percentages of moisture,

crude fat, crude protein, ash, crude fibre from 100 represents primarily the amount
of available carbohydrate substances.
 12

E. Physico-chemical properties of lipids

1. Specific gravity of fats and oils
The oil or fat sample may be preserved under an atmosphere of nitrogen in
the cold. The sample should be allowed to reach the room temperature and mixed
well before being subjected to the various analytical tests.
The specific gravity of a liquid is the weight of a given volume of liquid at
the specified temperature, compared with the weight of an equal volume of water at
the same temperature, all weighings being taken in air.
The specific gravity bottle (or pycnometer) is weighed empty (WB)
The bottle is then filled completely with the liquid and weighted (WL).
After cleaning, the bottle is filled completely with distilled water and
weighed (Ww).
The temperature of the liquid is noted.
WL - WB
Specific gravity =
Ww - WB
Note : The temperature at which the specific gravity is determined should
be specified.

2. Refractive index

The refractive index (n) of a substance is the ratio of the velocity of light in
vacuum to its velocity in the substance. It varies with the wavelength of light used
in its measurements. It may also be defined as the ratio of the sine of angle of
incidence to the sine of the angle of refraction.
Clean the refractometer with alcohol and ether -
A drop of oil or fat (in case of a solid fat the temperature should be suitably
adjusted by circulating hot water) is placed on the prism. The prism is closed by the
ground glass-half of the instrument. The dispersion screw is adjusted so that no
colour line appears between the dark and illuminated halves. The dark line is
adjusted exactly on the cross wires and the refractive index is read on the scale.
 13

Usually commercial instruments are constructed for use with white light but
are calibrated to give the refractive index in terms of sodium light of wavelength,
589.3 nm at a temperature of 20°C unless otherwise specified.

3. Acid value

The acid value of a fat is the number of mg of KOH required to neutralise

the free acid in 1 g of the substance.
Reagents
1. A mixture of equal volume of alcohol (95%) and ether
2. 1% phenolphthalein in alcohol.
3. 0.1 N KOH

Procedure
About 10 g of the oil or fat is weighed accurately into a 250 ml conical flask
to which 50 ml of a mixture of equal volume of alcohol and ether previously
neutralised is added after the addition of 1 ml of phenolphthalein solution. If
necessary, the contents may be warmed in a water bath until the oil is completely
dissolved. The solution is titrated with N/10 KOH with constant shaking until a
pink colour persists for 15 sec. The titre value in ml (a) is noted.
a x 0.00561 x 1000
Acid value =
Wt (g) of substance
If the normality of KOH is not exactly 0.1 N and if it is N then the above
equation is multiplied by the factor N / 0.1

4. Saponification value

Reagents
1. KOH solution : 35 to 40 g of KOH pellets are dissolved in 20 ml of water
to which sufficient alcohol (95%) is added to make 1 litre. The solution is
 14

allowed to stand overnight and the clear supernatant is used for the
estimation.
2. 0.5 N HCl.
3. 1% Phenolphthalein solution in 95% alcohol.
Procedure
About 2 g of the substance is weighed accurately in a 250 ml round bottom
flask. Twenty five ml of the alcoholic KOH solution is added, a reflux air
condenser is attached and the flask is kept in a boiling water bath for 1 hr. The
contents of the flask are frequently mixed. While the solution is still hot, 1 ml of
phenolphthalein solution is added and the excess alkali is titrated against 0.5 N HCl
(a). The experiment is repeated without the oil or fat to obtain the blank value (b).
Since 1 ml of 0.5 N HCl is equivalent to 0.02805 g of KOH, the following equation
is used to calculate the saponification value.

(b-a) x 0.02805 x 1000

Saponification value =
Wt (g)of substance
Note: If the HCl has a normality of ‘N’ then the above equation is multiplied by a
factor of : N
0.5

5. Iodine value
The iodine value of a substance is the weight of iodine absorbed by 100
parts by weight of the substance.

Reagents
1. Iodine monochloride solution : 5 ml iodine monochloride is dissolved in 500
ml glacial acetic acid.
If iodine monochloride is not readily available, the solution may be prepared
by dissolving 8 g Iodine trichloride in about 200 ml of glacial acetic acid with
heating in a water bath under moisture free condition (stoppered with a tube
containing fused calcium chloride). Nine g of iodine is taken into another flask and
dissolved in 300 ml of carbon tetrachloride. The two solutions are mixed and
sufficient glacial acetic acid is added to make the volume 1 litre.
 15

2. 0.1 N sodium thiosulphate solution.

3. 15 % Potassium iodide solution.
4. Starch mucilage.
Procedure
The substance is accurately weighed into a dry iodine flask and is dissolved
by the addition of 10 ml of CCl4 (or CHCl3). Twenty ml of iodine monochloride
solution is then added. The flask is stoppered with a stopper previously moistened
with Kl solution and the mixture is allowed to stand in the dark for 30 min. at a
temperature between 15-25oC. Fifteen ml of Kl solution is pipetted into the cup
top, the stopper is carefully removed and rinsed along with the sides of the flask
with 100 ml of water. The flask is then shaken and the contents are titrated with 0.1
N Na2S2O3 solution using starch mucilage as indicator towards the end of the
titration (starch is added only when the colour of the reaction mixture is faint
yellow). The volume of thiosulphate required (a) is noted. Simultaneously, the
experiment is carried out without the oil or fat to get the blank value (b).

(b-a) x 0.01269 x 100

Iodine value =
Wt (g) of substance

If the normality of the Na2S2O3 solution is N, the above equation is

multiplied by the factor N

0.1

The approximate weight (in g) of the original substance, if needed, may be

calculated by dividing the figure 20 by the highest expected iodine value. eg.
expected iodine value of an oil is 80, then the amount to be taken for analysis will
be 20/80 = 0.250 g
 16

F. Carbohydrate content

1. Determination of total carbohydrate

(with anthrone reagent)

Principle
Carbohydrates are hydrolysed by boiling with dilute HCl to
monosaccharides which are dehydrated by conc. H2SO4 to furfural or derivatives. It
then reacts with anthrone to produce a green color with absorption maximum at 620
nm. The method is useful to determine total carbohydrates that include reducing
and nonreducing sugars and starch. Cellulose is not hydrolyzed and is not
determined by this method.
Reagent :
1. 2 N HCl : Dilute 90 ml of conc. HCl into distilled water
and volume is made upto 500 ml.
2. Anthrone Reagent : Prepare fresh by dissolving 0.20 g anthrone in
100 ml of chilled conc. H2SO4.
3. Standard glucose : Prepare stock solution containing 100 mg
solution glucose in 100 ml aqueous solution. Dilute 10
ml of it to 100 ml with water to set the working
solution.
Procedure :
Take 100 mg of a thoroughly ground dry sample in a boiling tube containing
6 ml of 2 N HCl and place it in boiling water for at least 3 hours. Cool and add 50
ml of water, centrifuge and dilute the supernatant to 100 ml. Take 0.5 ml and 1 ml
aliquots in test tubes in duplicate. Also take 0, 0.1, 0.3, 0.5, 0.7 and 0.9 ml of the
working standard glucose solution in duplicate. Raise volume to 1 ml in standard as
well as sample tubes with water and place in ice bath. Add 4 ml of chilled anthrone
reagent to each tube and mix well. Place the tubes in boiling water for 10 minutes.
Blue-green color will appear. Cool the tubes under tap water and read the
absorbance at 620 nm. Draw a standard curve by plotting amount of glucose (g) on
X-axis vs. absorbance on the Y-axis. From the standard curve find the amount of
 17

carbohydrate (glucose) say Y g corresponding to the absorbance value of the

sample.
Calculations :
 X ml of sample contains Y g of carbohydrate.
 100 ml (i.e. 100 mg) sample contains.

Y g Y 100
= x 100 g = x mg carbohydrate in 100 mg sample
X ml X 1000

= Y/X mg carbohydrate/g sample.

 18

2. Determination of starch content

Principle
Finely powdered cereals and pulses are treated repeatedly with hot 80%
alcohol. The residue rich in starch is solubilised with perchloric acid. Filtered
perchloric acid extract is treated with anthrone-sulphuric acid to determine glucose.
Reagents:
1. Glucose standard : Dissolve 0.1 g of anhydrous glucose in 100 ml of water
containing 0.1% benzoate. Dilute 10 ml of stock solution to 1 litre and use 5 ml
for the standardisation. Prepare the diluted standard fresh.
2. Anthrone sulphuric acid : Dissolve 2 g anthrone in 1 litre of cold 95% H2SO4.
Store at 4oC and prepare fresh every 2 days.
3. Ethyl alcohol-water : Dilute 1.68 litre of 95% ethyl alcohol to 2 litre with water.
4. Perchloric acid : Add 270 ml of 72% perchloric acid to 100 ml of water. Store
in glass stoppered container.
Procedure
Extraction of sugars and starch : Weigh 0.2 g of flour into a 50 ml
centrifuge tube. Add a few drops of 80% alcohol to wet the flour and prevent
clumping, add 5 ml of water and stir thoroughly. Add 25 ml of hot 80% ethyl
alcohol, mix well and centrifuge after 5 min. of standing. Decant and discard the
alcoholic solution. Add 30 ml of fresh hot 80% ethyl alcohol, stir and centrifuge as
before. Discard the alcoholic solution. Repeat this washing twice for a total of 4
washings or until a test with anthrone is negative.
To the residue after final centrifugation add 5 ml of water. Cool in ice water
and while stirring add 6.5 ml of diluted perchloric acid reagent. Stir for about 5 min
with a glass rod. Keep for 15 min by stirring occasionally. Add 20 ml of water and
centrifuge. Pour the aqueous starch solution into a 100 ml volumetric flask cooled
in ice water and stir while adding 6.5 ml of diluted perchloric acid reagent.
Solubilise as before for 30 min at 0oC with occasional stirring and wash the
contents of the tube into a 100 ml flask containing the first extract. Dilute the
combined solutions to 100 ml and filter.
Determination of starch : Dilute 5 to 10 ml of the filtered starch solution
to 500 ml or to contain, 25 to 100 g of starch per 5 ml of solution. Pipette 5 ml of
 19

the diluted solution into a test tube, cool in a water bath and add 10 ml fresh
anthrone reagent. After the anthrone reagent has been added to all the tubes, mix
each one thoroughly and heat them together for 7.5 min. Read the colour in a
photocolorimeter at 630 nm.
Prepare a standard curve every time using 0.50 and 100 g glucose
containing the same amount of perchloric acid as that in starch aliquots and use this
calibrated curve to obtain the yield of glucose from starch. Multiply glucose value
by 0.9 to convert into starch value.
 20

G. Amino acid and protein content

1. Determination of protein content

Lowry’s procedure
Principle
The final colour is a result of, (a) biuret reaction of protein with copper ion
in alkali and (b) reduction of the phosphomolybdic-phosphotungstic reagent by the
tyrosine and tryptophan present in the treated protein.
Reagents :
1. 4% Na2CO3
2. 0.5% CuSO4.5H2O in 1% potassium sodium tartarate
3. Alkaline copper solution: Mix 50 ml of reagent 1 with 2 ml of reagent 2
(prepare fresh every day).
4. 0.1 N NaOH
5. Diluted Folins’ reagent : Dilute Folin-Ciocalteau reagent (purchased from
V.P. Chest Institute, Delhi) with an equal volume of 0.1 N NaOH.
6. Standard protein solution : The standard solution is prepared with bovine
serum albumin to have a concentration of 100 g protein/ml.
Procedure
The test sample containing about 50-100 g protein/ml is taken at three
levels, 0.5, 1.0 and 1.5 ml. The necessary amount of 0.1 N NaOH is added to bring
the volume to 1.5 ml. To this 1.5 ml. of reagent 3 is added. Each tube is shaken
and allowed to stand for 10 min after which exactly 0.15 ml. of diluted Folin’s
reagent added with continuous shaking. The tubes are allowed to stand for half an
hr and then read at a wavelength of 750 nm. For more concentrated solutions, the
readings may be kept in a workable range by reading it at a wavelength of 500 nm.
The unknown sample is then read off. The standard curve and the necessary
calculations are made.
 21

2. Determination of available lysine

Principle
The procedure involves conversion of lysine residues with reactive epsilon
amino groups in the food proteins, into a yellow epsilon-dinitrophenyllysine by
treatment of the material with fluorodinitrobenzene, followed by acid hydrolysis,
Ether soluble interfering compounds are removed by extraction and the extinction
of the residual aqueous layer is measured. A blank value is obtained by treatment
with methoxycarbonylchloride and extraction of the ether soluble lysine compound.
Reagents
1. 10% NaOH.
2. Buffer, pH 8.5 : Mix 8% NaHCO3, 8% Na2CO3, 19:1 (V/V) adjust pH with
NaOH or HCl as the case may be.
3. HCl : 8.1N and 1N
4. Fluorodinitrobenzene (FDNB) : 2.5 % (V/V) solution in ethanol (to be
prepared freshly).
5. Dinitrophenyllysine hydrochloride (DNPL-HCl. H2O): Prepare a stock
solution having 1 mg DNPL-HCl/ml in 1 N HCl. Working standard is made
daily by diluting 1 ml of the stock standard solution to 100 ml in 1N HCl.
Procedure
The material is first defatted and ground and the nitrogen is estimated by
micro-Kjeldahl method. The amount of the sample weighed should be such that it
contains about 30-50 mg nitrogen. The sample is taken in a 250 ml round bottom
flask to which 10 ml of 8 % NaHCO3 solution is added, the content is mixed by
gentle swirling and then left to stand for 10 min. Add 0.3 ml of FDNB reagent to
the flask. The contents of the flask are then thoroughly mixed and the flasks are
covered with black paper and shaken for 2 hr on a shaker. The alcohol is then
removed by evaporation on a boiling water bath. The residue is extracted with four,
50 ml portions of anhydrous, peroxide free ether or till the ether washing is
colourless. Each time ether is removed by decantation. The residual ether is
removed by aeration. Add 24 ml of 8.1 N HCl to the flask and the resulting mixture
refluxed with gentle boiling for 16 hr. One or two glass beads are added to prevent
bumping. The flasks are then ice cooled for 1-2 hr and filtered through a Whatman
No. 41 filter paper in a 250 ml volumetric flask. The volume of the filtrate and
 22

washings are made up to the mark with distilled water. Two ml of the filtrate are
pipetted into two 10 ml glass stoppered test tubes marked A and B and also into a
conical flask marked C. The contents of the tubes A and B are extracted twice with
5 ml of ether. The ether layer is discarded and the tube held in a boiling water bath
until the effervescence ceases. The tubes are then allowed to cool to room
temperature. Tube A is made upto the 10 ml mark with 1 N HCl and kept for the
final readings. The contents of flask C are then titrated against 10% NaOH using
phenolphthalein as the indicator and the contents of the flask discarded and the
volume of NaOH used is noted. The same volume of NaOH is then added to tube
B, followed by 2 ml of buffer solution. Methoxycarbonylchloride (0.05 ml) is then
added and the tubes are shaken vigorously in order to disperse and dissolve the
compound. (The extraction with methoxycarbonylchloride is done only to obtain a
blank value for non-lysine interfering colour). After 5-10 min 0.75 ml of
concentrated HCl is added cautiously to prevent the contents from frothing.
The contents are then extracted twice with 50 ml portions of ether. The
ether washing are discarded and the residual ether is evaporated over a boiling water
bath. The tubes are allowed to cool and then the volume is made upto 10 ml with
distilled water. The OD of the contents of tubes A and B is measured in a
photoelectric colorimeter at 420 nm. Reading A minus B is taken as the extinction
due to epsilon DNP-lysine, the concentration of which is extrapolated from the
standard graph obtained by using concentrations of standard DNP-lysine solution.
Calculation
Available lysine is expressed as g available lysine/100 g protein =
0.851 x 0.4682 x dil. factor x 100 x 100 x conc. of DNP-HCl. H2O
Wt. of sample x % protein
Mol. wt. of epsilon DNP-lysine = 312.3
Mol. wt. of epsilon DNP-lysine HCl. H2O = 366.8
312.3
Conversion of epsilon DNP-lysine HCl. H2O to epsilon DNP-lysine = = 0.851
366.8

146.19
Conversion of epsilon DNP-lysine to lysine = = 0.4682
312.3
 23

5. Vitamin content of food

1. Determination of -carotene
Principle
The individual carotenoids are separated on a column of calcium hydroxide
or alumina and determined spectrophotometrically. The values for their respective
vitamin A potency are used to arrive at the total vitamin A value of the foodstuff.
Procedure:
Preparation of the sample : Twenty five g of freshly ground sample is
allowed to stand overnight in a mixture of 100 ml of petroleum ether-acetone (1:1).
The extract is then filtered and the residue on the filter is washed twice with
successive 500 ml portions of the mixture till all the yellow colour is extracted. The
pooled filtrate is shaken with 50 ml portions of water and water washings are
discarded. This is repeated twice. The solvent layer is then dried over anhydrous
sodium sulphate and concentrated under reduced pressure to a final volume of 4 ml.
Separation of pigments using calcium hydroxide column : A column of
30 x 1 cm is packed with 6 g of calcium hydroxide under gentle suction. About 1-2
g of anhydrous sodium sulphate is placed on top of the packed column. The column
is initially wet by passing petroleum ether. Two ml of the concentrated extract
containing about 40-100 g of the total carotene is charged on to the column. Light
suction is employed and a 1% acetone in petroleum ether is used as developing
solvent. The separated bands are individually eluted and their spectra studied in a
Beckman DU spectrophotometer to identify the different carotene fractions.
For quantitative estimation of different carotene fractions, standard graphs
are obtained with graded concentrations of pure -carotene dissolved in petroleum
ether and this curve is used for determining the concentrations of the solutions of
pigments whose main absorption is around 460 nm.
Separation using alumina column : Two ml of the concentrated carotene
extract is loaded on to a column of alumina (10 x 1 cm) containing 3% anhydrous
sodium sulphate and eluted with petroleum ether. The -carotene is now eluted
with petroleum ether containing 3% acetone. The volume of this eluate is made
 24

upto 5 ml and OD of this eluate is measured at 450 nm in Beckman DU

spectrophotometer.
Calculation
1 OD is equivalent to 4 g/ml of -carotene when measured in 1 cm cell.
The relative potencies of different carotenoids reported in the literature are
used to calculate the vitamin A value in the case of each carotenoid fraction. The
sum of the values of all the fractions present in the chromatogram of a particular
foodstuff gives its true vitamin A value.

2. Determination of ascorbic acid content

Dye method

Principle
The blue colour produced by the reduction of 2, 6 - dichlorophenol
indophenol by ascorbic acid is estimated colorimetrically.

Reagents
6. Acetate buffer, pH 4.0 : 300 g of anhydrous sodium acetate, 700 ml of water
and 1 litre of glacial acetic acid are mixed.
7. Dye solution : 25 g of the sodium salt of 2.6 - dichlorophenol indophenol is
dissolved in distilled water and volume made upto 200 ml.
8. 6% Metaphosphoric acid (HPO3).
9. Ascorbic acid standard (1 mg per ml): 100 mg of pure ascorbic acid is
dissolved in 100 ml of 6% HPO3.
Procedure
A weighed quantity of the material is blended with a convenient volume of
6% HPO3 and the slurry is diluted to obtain a final mixture containing
approximately 20 g of ascorbic acid per ml (for example, in case of leaves, 5 g of
the sample can be blended with 6% HPO3 to make 50 ml and 5 ml of the slurry is
further diluted to 50 ml).
The mixture is then filtered and 2-5 ml of the filtrate is placed in a 50 ml
separating funnel (A). The same amount of the extract (6% HPO3) is taken in two
more separating funnels, B and C. Funnel B serves as the dye blank, and to funnel
 25

C which serves as a standard 0.1 ml (equivalent to 0.1 mg ascorbic acid) of the

ascorbic acid standard solution is added. An amount of acetate buffer equal to the
volume of the extract taken is then added to all the three funnels, followed by 2 ml
of the dye solution. Xylene 10 ml is then added quickly and the contents shaken for
6-10 sec. After the layers are separated, the lower water layer is removed and the
colour in the xylene extract is measured in a photoelectric colorimeter at 500 nm.
Calculation
The calculation for the ascorbic acid content is based on the reduction in the
OD of the dye solution on reaction with ascorbic acid. If the ODs of the extracts
from funnels A, B and C are a, b and c, then the ascorbic acid contained in the
amount of the extract taken for reaction with the dye is equal to 0.1 (b –a) / b-c
mg. The ascorbic acid content of the material can then be calculated by applying
the necessary dilution factors.
Note:
Reagent blank with the addition of the dye should always be run along with
the sample.

3. Determination of Niacin

Principle
Nicotinic acid reacts with cyanogen bromide and aromatic amines like
aniline, metol etc., to give a yellow coloured compound, which is measured
colorimetrically.
Reagents
1. Stock calcium hydroxide suspension : 250 g of calcium hydroxide in 1 litre
of water.
2. Working calcium hydroxide suspension : Reagent 1 is suitably diluted to
give 2 N calcium hydroxide. This is checked by titration.
3. Zinc sulphate solution : 80 g of ZnSO4. 7 H2O is dissolved in 100 ml of
water.
4. Phosphate solution : 5 g of KH2PO4 is dissolved in 100 ml of water.

5. Niacin standard : 500 mg niacin is dissolved in water and 5 ml of 10 N

H2SO4 is added and then the volume is made upto 500 ml.
 26

6. Cyanogen bromide solution : 27 ml of bromine kept in a conical flask

immersed in ice is neutralised with 10% solution of freshly prepared NaCN
using a burette till the solution becomes colourless. Ten extra drops of
NaCN are added and the solution made upto 1 litre .
7. Buffered CNBr : 500 ml of CNBr solution (6) are added to 25 g of KH2PO4
in a 500 ml volumetric flask, dissolved and kept in the cold.
8. 8% metol in 0.5 N HCl.
Procedure
Digestion of sample : Two g of the finely powdered sample is suspended in
10 ml of calcium hydroxide and the volume is made upto 50 ml.
Sample, recovery and blank tubes are similarly prepared and all of them are
heated in a boiling water bath for 90 min. The contents are stirred frequently in the
early stages. After 90 minutes the tubes are cooled in a bath of cold water and the
volume of the solution is made up again to 50 ml and centrifuged for 20 minutes at
2000 rpm. A 25 ml aliquot of the centrifugate is transferred to another calibrated
tube and acidified with 0.5 ml of 8 N HCl and then 2 ml of ZnSO4, solution 2 drops
of caprylic alcohol and 2 drops of phenolphthalein are added. Add 4 N NaOH with
cooling till Zn (OH)2 is formed. Then 1 N NaOH is added until the mixture turns
pink. Add 5 N H2SO4 till the pink colour just disappears after that the volume is
made upto 50 ml, centrifuged and aliquots of centrifugate are used to develop the
colour.
Development of colour : Each solution is taken in 4 tubes (A, B, C, D) to
allow for corrections due to amine, CNBr and other interfering colours.
A B C D
ml ml ml ml

Filtrate 5 5 5 5

CNBr reagent 2 - 2 -

5% KH2PO4 - 2 - 2

8% metol in 0.5 N HCl 3 3 - -

0.5 N HCl - - 3 3
The yellow colour developed is read at 400 nm in a colorimeter.
 27

Calculation
The colour due to the 5 ml filtrate (0.1 g sample) alone = A- (B + C-D) = a.
The colour in the recovery tube(0.1g sample + 5g niacin) = AR - (BR+CR - DR) = b
b-a 5a
b-a = 5 g of niacin or 1g of niacin = and 0.1 g of substance contains
5 b-a

5a 100
 % niacin = x
(b - a) 0.1
 28

I. Mineral content of food

1. Preparation of ash solution
The ash is moistened with a small amount of glass distilled water (0.5
- 1.0 ml) and 5 ml of distilled hydrochloric acid is added to it. The mixture is
evaporated to dryness on a boiling water bath. Another 5 ml of hydrochloric acid is
added again and the solution evaporated to dryness as before. Four ml of
hydrochloric acid and a few ml of water are then added and the solution is warmed
over a boiling water bath and filtered into a 100 ml volumetric flask using Whatman
No 40 filter paper. After cooling, the volume is made upto 100 ml and suitable
aliquots are used for the estimation of phosphorus, iron and calcium.

1. Extraction of minerals

Principle
Liquid sample can be used as such whereas solid samples of food are
digested with acid to oxidize the organic material and to extract minerals in an
aqueous solution before analysis for minerals.
Materials :
Concentrated sulfuric, nitric and perchloric acids, deionized or glass distilled
water, Kjeldahl flask (100 ml), pipettes, table centrifuge etc.
Procedure
Take 25 ml of a 3 : 2 : 1 (v/v) mixture of concentrated acids viz. nitric,
perchloric and sulfuric acid in a 100 ml Kjeldahl flask and add 2-3 gm of finely
powdered dry food sample in it. Leave for 4 to 12 hours in a fume chamber. Heat
slowly for 30 minutes and later strongly till the acidic solution is clear and no more
fumes come out. It may take 4 hours or more. About 5 ml of nitric acid may be
added if contents of the flask seems to become dry followed by further heating.
Cool and wash the contents of flask four times with 2 ml aliquots of water (use
deionized water only) to ensure complete transfer of minerals into a tube.
Centrifuge at 5000 rpm for 30 minutes. Transfer the clear supernatant in a graduated
(15 ml) tube and make the volume 10 ml with water. The solution can be stored in
acid washed polythene bottles and used for determination of minerals.
 29

3. Determination of Calcium content

Titrimetric method
Principle
Calcium is precipitated as oxalate and is titrated with standard potassium
permanganate.
Reagents
1. 4% ammonium oxalate solution.
2. Dilute ammonia solution (2 ml of liquor ammonia + 98 ml water)
3. 1 N H2SO4
4. 0.01 N Potassium permanganate solution.
5. 0.01 N Oxalic acid : Sodium oxalate is dried in an oven at 100 - 105oC for 12
hr. Exactly 0.67 g is dissolved in redistilled water, 5 ml concentrated H2SO4 is
added and solution is made upto 1 litre after it has cooled down.
Standardisation of potassium permanganate solution : 25 ml of 0.01 N oxalic
acid is transferred to an Erlenmeyer flask. One ml of concentrated H2SO4 is added,
warmed to about 70oC and titrated against KMnO4 solution, till the faint pink colour
remains.
Procedure
Two ml of mineral solution is taken into a 15 ml centrifuge tube. Add 2 ml
of distilled water and 1 ml of 4% ammonium oxalate solution and mix thoroughly
and leave overnight. Again the contents are mixed and centrifuged for 5 min. at
1500 rpm. The supernatant liquid is poured off and the centrifuge tube drained by
inverting the tube for 5 min. on a rack (care should be taken not to disturb the
precipitate). The mouth of the centrifuge tube is wiped with a piece of filter paper.
The precipitate is stirred and the sides of the tubes are washed with 3 ml of dilute
ammonia. It is centrifuged again and drained as before. The precipitate is washed
once more with dilute ammonia to ensure the complete removal of ammonium
oxalate. The precipitate is dissolved in 2 ml of 1 N H2SO4. The tube is heated by
placing it in a boiling water bath for 1 min and titrated against 0.01 N KMnO4
solution to a definite pink colour persisting for at least 1 min.
 30

Calculation
1 ml of 0.01 N KMnO4 is equivalent to 0.2004 mg of calcium.
100
mg of calcium/100 ml solution = (X-b) x 0.2004 x
2
where,

X = number of ml of 0.01 N KMnO4 required to titrate the sample,

b = number of ml of 0.01 N KMnO4) required to titrate 2 ml of H2SO4
(blank).

If the normality of KMnO4 is N, the value obtained in the above formula

should be multiplied by the factor, N
0.01
Note : Potassium permanganate solution needs to be frequently standardised.

4. Iron content of food

Wong’s method

Principle
Iron is determined colorimetrically making use of the fact that ferric iron
gives a blood red colour with potassium thiocyanate.
Reagents
1. 30% H2SO4
2. 7 % Potassium persulphate solution : 7 g potassium persulphate is dissolved
in glass distilled water and the solution is made upto 100 ml.
3. 40% Potassium thiocyanate solution : 40 g KCNS is dissolved in 90 ml glass
distilled water, 4 ml acetone added and the volume made upto 100 ml.
2. Standard iron solution : 702.2 mg ferrous ammonium sulphate is dissolved in
100 ml glass distilled water and after addition of 5 ml of 1:1 HCl, the solution
is made upto 1 litre and mixed thoroughly (0.1 mg iron / ml). The standard
solution is prepared fresh once in 6 months.
 31

Working standard solution (10 g iron / ml) is prepared by diluting the above
solution 10 fold.
Procedure
To an aliquot (6.5 ml or less) of the mineral solution enough water is added
(if necessary) to make upto a volume of 6.5 ml followed by 1.0 ml of 30% H2SO4,
1.0 ml potassium persulphate solution and 1.5 ml 40% KCNS solution. The red
colour that develops is measured within 20 min. at 540 nm.
Haemoglobin iron
Two ml of conc. H2SO4 is taken in a 50 ml volumetric flask. Add exactly
0.5 ml of well mixed blood, mix and add 2 ml potassium persulphate, agitate the
flask, cool and dilute with about 25 ml distilled water. Then 2 ml of sodium
tungstate is added and the volume made upto the mark. Filter using Whatman
No.42 filter paper. Transfer 15 ml of the filtrate to a fresh tube, add 1 ml of
potassium persulphate and 4 ml of potassium thiocyanate. Mix and read the colour
at 540 nm in a colorimeter. A standard (10-100 g) is run similarly and a standard
graph is prepared.
Calculation
Haemoglobin (g) = 0.3 x mg iron
Note : If use of reagent containing traces of iron cannot be avoided, it should be
seen that the final solutions of standards and test contain identical quantities of
these reagents. Potassium thiocyanate should be added just before taking the
readings.

5. Determination of Magnesium content

Principle
Magnesium is converted to magnesium pyrophosphate, which is estimated
gravimetrically.
Reagents
1. 10% Ammonium phosphate solution.
2. 10% Sodium citrate solution.
3. 0.1% Methyl red indicator.
4. 1 : 4 and 1: 10 : : ammonia : water solution.
 32

Procedure
Thirty ml of conc. HNO3 is added to the calcium free filtrate (obtained from
the filtrate after precipitation of calcium as oxalate) and the solution is evaporated
completely on a boiling water bath. Five ml of conc. HCl and 100 ml of water are
then added and the solution is stirred well with a glass rod. It is followed by the
addition of 10 ml of 10% ammonium phosphate solution and 5 ml of 10% sodium
citrate solution and the mixture is stirred. After adding 2 or 3 drops of methyl red
indicator the solution is neutralised with the addition of 1:4 dilute ammonia. Strong
ammonia (25 ml) is then added, stirred vigorously and the mixture is left to stand
overnight, filtered through Whatman No. 40 or 44 filter paper and washed free from
chlorides using 1:10 dilute ammonia (tested with HNO3 + silver nitrate solution).
The funnel with the precipitate on the filter paper, is dried in an oven. The filter
paper is then transferred to a weighed crucible (the crucible is previously heated,
cooled and weighed) and ashed slowly over a burner. It is then kept in a muffle
furnace at 900oC for 2 hr. The crucible and the contents are cooled in a desiccator
and weighed to get magnesium as its pyrophosphate.
Calculation
Magnesium (mg) /100 g of sample =
48.64 100 100
weight of ash x x x x 1000
222.60 ash solution taken Wt of sample
for estimation

6. Determination of Sulphur content

Principle
Sulphur is precipitated with barium chloride and is estimated
gravimetrically.
Reagents
1. 0.1% Methyl orange
2. 40% NaOH
3. 5% Barium chloride
 33

Procedure
Five ml of conc. HNO3 is added to 2-3 g of sample weighed into a micro-
Kjeldahl flask and the flask is covered with glass stopper. It is kept in a boiling
water bath for 10 hr and then on a sand bath and is digested on a low flame till a
liquid digest results (additional nitric acid is added if necessary). Two ml of
perchloric acid is added to this solution and the temperature is raised. Addition of
nitric acid and perchloric acid are repeated till the digest turns into a clear solution.
Digestion is continued for a further 10-16 hr. By this time all the nitric acid would
have escaped and the remaining clear solution can be transferred into a 500 ml
beaker by repeated washings with distilled water to make approximately 200 ml.
One or two drops of methyl orange indicator are added and the solution is
neutralised with the addition of 40% NaOH. The solution is again acidified with
HCl and boiled for 5 min. Ten ml of 5% barium chloride is added and the solution
is boiled for 5 min. It is kept overnight and then filtered through Whatman No. 40
or 44 filter paper and washed with distilled water till free from barium chloride.
The precipitate along with the filter paper is kept in an oven for drying. The filter is
then taken in a weighed crucible and ignited, taking care to avoid spurting and
finally ashed at 600oC for 3-4 hr and weighed.
Calculation
100
Sulphur (g) / 100 of sample = Wt of the ash x 0.1374 x
Wt of sample
 34

J. Miscellaneous
1. Determination of oxalic acid
Principle
The oxalic acid is extracted from foodstuffs and precipitated as calcium
oxalate which is then titrated against standard potassium permanganate.
Reagents
1. 2 N HCl.
2. Phosphoric-tungstate reagent : Forty ml of phosphoric acid is added to 24 g
of sodium tungstate dissolved in water and the solution is diluted to 1 litre
with water.
3. Calcium chloride buffer : 25 g calcium chloride is dissolved in 500 ml of
50% glacial acetic acid and 330 g of sodium acetate is dissolved in 500 ml
of water. Both the solutions are then mixed well.
4. 0.1% Methyl red.
5. N/100 KMnO4
Procedure
Extraction of the sample : Five g of well ground sample is added into 100
ml of 2 N HCl and the mixture is shaken well for about 2 hr in a mechanical shaker.
It is then centrifuged and filtered (in the case of fresh sample, 10 g of the sample is
well ground in a mortar along with 25 ml of 2 N HCl). The mixture is transferred to
the same beaker and weighed. It is then boiled for about 15 min and cooled. The
mixture is adjusted to the previous weight with distilled water, made upto 100 ml
with 2 N HCl, shaken well and filtered.
Twentyfive ml of the filtrate is added to 5 ml phosphoric-tungstate reagent,
stirred well and kept overnight. The next day, it is centrifuged and filtered. To 20
ml of the filtrate is added 2 or 3 drops of methyl red, neutralised with ammonia, 5
ml of calcium chloride buffer is added and stirred well. The mixture is allowed to
stand overnight. At the end, it is filtered through Whatman No. 40 or 44 filter paper
and washed free from chloride using distilled water (silver nitrate test). The
precipitate along with the filter paper is transferred to the same beaker and some
distilled water is added followed by 5 ml of 2 N H2SO4. The mixture is heated to
80oC over a burner and titrated against N/100 potassium permanganate solution.
 35

Calculation:
1 ml of N/100 KMnO4 = 0.45 mg of oxalic acid.

2. Determination of trypsin inhibitor

Principle
The activity of the enzyme trypsin is assayed using casein as substrate.
Inhibition of this activity is measured in the extracts.
Reagents
1. 0.1 M Sodium phosphate buffer, pH 7.5
2. 2% Casein solution in phosphate buffer.
3. Trypsin (E. Merck) solution, 5 mg/ml
4. 0.001 M HCl
5. 5% TCA

Procedure
Extraction of samples : Four g of the pulverised defatted plant material is
treated with 40 ml of 0.05 M sodium phosphate buffer(pH 7.5) and 40 ml of
distilled water. The samples are shaken for 3 hr and then centrifuged at 700 g for
30 min at 15oC. The supernatants are diluted in such a way that there is an
inhibition between 40 and 60 % of the control enzyme activity.
The incubation mixture consists of 0.5 ml of trypsin solution, 2 ml of 2 %
casein, 1.0 ml of sodium phosphate buffer (pH 7.5, 0.1 M), 0.4 ml HCl (0.001 M)
and 0.1 ml extract. In all the cases the total volume of the incubation mixture is
kept as 4 ml. Incubations are carried out of 37oC for 20 minutes after which 6.0 ml
of 5% TCA solution is added to stop the reaction and corresponding blanks are run
concurrently. In this method, one trypsin unit (TU) is arbitrarily defined as an
increase of 0.01 absorbance unit at 280 nm in 20 min for 10 ml reaction mixture
under the conditions described and the trypsin inhibitory activity as number of
trypsin units inhibited (TUI).
Note : In order to express the values as specific activities, the total TUI has to be
expressed per mg protein. The protein content is generally estimated by Lowry’s
 36

method. The values for trypsin inhibitory activity must have about 40-60%
inhibition in order to have a reproducible and accurate result.

3. -N-oxalylamino-L-alanine

Principle
-N-Oxalylamino-L-alanine (BOAA) is a neurotoxic amino acid found in
Lathyrus sativus. It reacts with ninhydrin giving a violet colour, the intensity of
which is read in a colorimeter.
Reagnets :
1. 70% aqueous ethanol
2. Buffer system( pH 3.6) : Acetic acid : Pyridine : Water : : 5.0 : 0.5 : 94.5
3. 0.1% Ninhydrin in acetone
4. Copper sulphate-ethyl alcohol solution : 300 mg of CuSO4. 5 H2O and 50
ml distilled water are mixed with distilled ethyl alcohol just before elution
from paper.
5. Pure BOAA solution as standard.

Procedure
Extraction of seeds for BOAA estimation : Two g of powdered Lathyrus
sativus seeds are treated with 40 ml of 70% aqueous ethanol, stirred at intervals and
kept overnight. The contents are centrifuged and the residue is treated in the same
way once again. The supernatants are pooled and evaporated to dryness on a
boiling water bath at 100oC. The residue is dissolved in 10 ml of distilled water.
Analysis of BOAA : Fifty  l portions of the extract are applied on
Whatman No. 1 filter paper strips. Separation is carried out using a Beckman paper
electrophoresis cell (Durrum type, Model R) with a buffer system (pH 3.6) and a
voltage of 250 is applied for about 4 hr 30 minutes.
After the run, the strips are dried in an air oven at 80oC. The dry paper strips
are sprayed with 0.1% ninhydrin in acetone. They are air dried and the ninhydrin
spraying repeated. After air drying, the strips are heated at 80oC for 10 min.
 37

Standard BOAA solutions are spotted concurrently on separate paper strips and run
along with the samples. Matching with the position of the standard BOAA spot, the
corresponding test BOAA spots are eluted individually with 9 ml of mixture of
copper sulphate solution and ethyl alcohol. The extracted solution is read in Klett-
Summerson colorimeter using green filter (No. 54) and calculations are made after
comparison with standard BOAA curves.

Note: The mobility of BOAA under the specified conditions mentioned above is
about 6.7 cm towards anode and the next moving ninhydrin spot mobility is only
3.1 cm. By this method BOAA at a level of 0.0075  moles or 1.32 g could be
estimated. The per cent recovery of BOAA is found to be about 95% by this
method.
 38

APPENDIX - I

Relation between % transmittance and optical density

Transmi- O.D. Transmi O.D. Transmi O.D. Transmi O.D.

ttance -ttance -ttance -ttance
(%) (%) (%) (%)
100 0.000 75 0.125 50 0.301 25 0.602
99 0.004 74 0.131 49 0.310 24 0.620
98 0.009 73 0.137 48 0.319 23 0.638
97 0.013 72 0.143 47 0.328 22 0.658
96 0.018 71 0.149 46 0.337 21 0.678
95 0.022 70 0.155 45 0.347 20 0.699
94 0.027 69 0.161 44 0.357 19 0.721
93 0.032 68 0.168 43 0.367 18 0.745
92 0.036 67 0.174 42 0.377 17 0.770
91 0.041 66 0.181 41 0.387 16 0.796
90 0.046 65 0.187 40 0.398 15 0.824
89 0.051 64 0.194 39 0.409 14 0.854
88 0.056 63 0.201 38 0.420 13 0.886
87 0.061 62 0.208 37 0.432 12 0.921
86 0.066 61 0.215 36 0.444 11 0.989
85 0.071 60 0.222 35 0.456 10 1.000
84 0.076 59 0.229 34 0.469 9 1.046
83 0.081 58 0.237 33 0.482 8 1.097
82 0.086 57 0.244 32 0.495 7 1.155
81 0.092 56 0.252 31 0.509 6 1.222
80 0.097 55 0.260 30 0.523 5 1.301
79 0.102 54 0.268 29 0.538 4 1.398
78 0.108 53 0.276 28 0.552 3 1.523
77 0.114 52 0.284 27 0.569 2 1.699
76 0.119 51 0.292 26 0.585 1 2.000
 39

APPENDIX - II

Boiling points of some chemicals

The following boiling points are correct to the nearest degree centigrade.
o
Substance C

Acetone
... 56
Acetic acid ... 118
Amyl alcohol ... 130
Benzene ... 80
Butyl alcohol ... 118
Caprylic alcohol ... 180
Carbon disulphide ... 46
Chloroform ... 62
Ether ... 34
Ethyl alcohol ... 78
Hexane ... 68
Methyl alcohol ... 65
Toluene ... 111
Water ... 100
Xylene ... 138-144
 40

APPENDIX – III

Details of common acids and alkalies

Substance Specific gravity Percent Normality
(at room temperature) by weight
Ammonium hydroxide 0.90 28.0 15.1
(stronger)

Sodium hydroxide (saturated) 1.50 50.0 19.0

Sulfuric acid (concentrated) 1.84 96.0 35.9

Nitric acid (concentrated) 1.42 69.5 15.6

Hydrochloric acid 1.19 36.0 11.7

(concentrated)

Phosphoric acid (syrupy) 1.71 85.0 45.0

Acetic acid (glacial) 1.06 99.8 17.6

Perchloric acid 1.54 60.0 9.2

41
 APPENDIX - IV

Equivalent weight of common chemicals

Name Formula Mol. wt. Fraction Eq.

of Mol. Wt.
Wt.
Hydrochloric acid HCl 36.46 1.0 36.46

Sulphuric acid H2SO4 98.00 0.5 49.00

Oxalic acid (hydrated) (COOH)2 2H2O 126.05 0.5 63.03

Sodium hydroxide NaOH 40.00 1.0 40.00

Potassium hydroxide KOH 56.10 1.0 56.10

Sodium carbonate Na2CO3 106.00 0.5 53.00

(anhydrous)
Borax Na2B4O7.10H2O 381.44 0.5 190.72

Potassium permanganate KMnO4 158.00 0.2 31.60

Potassium dichromate K2Cr2O7 294.20 0.17 49.03

Mohr’s salt (Ferrous FeSO4 (NH4)2 392.10 1.0 392.10

ammonium sulphate) SO4 6H2O

Ferrous sulphate (crystals) FeSO4. 7H2O 278.00 1.0 278.00

Sodium oxalate (COONa)2 134.00 0.5 67.00

Ammonium oxalate (COONH4)2.H2O 144.08 0.5 72.04

Iodine I2 253.80 0.5 126.90

Hypo(Sodium Na2S2O3. 5H2O 248.20 1.0 248.20

thiosulphate)
 CONTENTS
Sl. No. Particulars Page No.

A. General directions to be taken in the laboratory 1

B. Food analysis 3
C. Sampling Procedure 3
D. Proximate analysis of food 6

1. Determination of moisture content 6

2. Determination of ash content 7

3. Determination of fat content 8

4. Determination of crude fibre content 9

5. Determination of crude protein content 10

6. Determination of carbohydrate content 11

E. Physico-chemical properties of lipids

1. Specific gravity of fats and oils 12

2. Refractive index 12

3. Acid value 13

4. Saponification value 14

5. Iodine value 14

F. Carbohydrate content

1. Determination of total carbohydrate 16

2. Determination of starch content 18

G. Amino acid and protein content

1. Determination of protein content 20

2. Determination of available lysine 21
 H. Vitamin content of food

1. Determination of -carotene 23

2. Determination of Ascorbic acid 24

3. Determination of Niacin 25

I. Mineral content of food

1. Preparation of ash solution 28

2. Extraction of minerals 28

3. Determination of calcium content 29

4. Determination of iron content 30

5. Determination of Magnesium content 31

6. Determination of Sulphur content 32

J. Miscellaneous

1. Determination of oxalic acid 34

2. Determination of trypsin inhibitor 35

3. Determination of -OAA 36

Appendix

I Relation between transmittance and optical

density

II Boiling points of chemicals

III Details of common acids and alkalies

IV Equivalent weight of common chemicals

45
 FOREWORD
The success of Indian Agriculture in recent past has put India from a position
of scarcity and dependence to the level of self- sufficiency and envisaged export potential.
Our achievements so far generally have been gratifying, but the war against malnutrition is
by no means over. Dietary inadequacies coupled with poor health care are reflected in
widespread growth retardation of children, and high incidence of low birth weight
deliveries, anaemia and iodine deficiency disorders. It is very essential to overcome these
problems by providing optimal nutritional status that can be achieved only by ensuring
habitual diets of families adequate with respect to all essential nutrients – not just calories.

Hence, time has come to bother much for assuring nutritional security to our
population in place of food security. For this one has to know the quality of food which is
determined by the level of nutrients (Carbohydrates, proteins, fats, vitamins, minerals and
water) and toxic substances present in the food. The nutrient composition is also affected by
different processing methods that need to be analysed. Then only , the right kind of food
products can be developed after the application of proper processing and fortification
techniques. Therefore a student or scientist engaged in the field of nutrition must have the
knowledge of the actual nutrient content and toxic substances of food, before and after the
application of processing methods.

The present manual on Food Analysis in which all the standardized

procedures have been mentioned in simple language will be helpful for analyzing the food
for its nutrient composition and toxic substances.

(V. P. Gupta)
 Dr. Usha Singh

DEPARTMENT OF FOOD & NUTRITION

COLLEGE OF HOME SCIENCE

2001
 Dr. V. P. Gupta
Vice-Chancellor
Phone (O): 91- 6274 - 74226
(R): 91- 6274 –74264/74377
Fax : 91- 6274 - 74255

FOREWORD
The success of Indian Agriculture in recent past has put India from a position
of scarcity and dependence to the level of self- sufficiency and envisaged export potential.
Our achievements so far generally have been gratifying, but the war against malnutrition is
by no means over. Dietary inadequacies coupled with poor health care are reflected in
widespread growth retardation of children, and high incidence of low birth weight
deliveries, anaemia and iodine deficiency disorders. It is very essential to overcome these
problems by providing optimal nutritional status that can be achieved only by ensuring
habitual diets of families adequate with respect to all essential nutrients – not just calories.

Hence, time has come to bother much for assuring nutritional security to our
population in place of food security. For this one has to know the quality of food which is
determined by the level of nutrients (Carbohydrates, proteins, fats, vitamins, minerals and
water) and toxic substances present in the food. The nutrient composition is also affected by
different processing methods that need to be analysed. Then only , the right kind of food
products can be developed after the application of proper processing and fortification
techniques. Therefore a student or scientist engaged in the field of nutrition must have the
knowledge of the actual nutrient content and toxic substances of food, before and after the
application of processing methods.

The present manual on Food Analysis in which all the standardized

procedures have been mentioned in simple language will be helpful for analyzing the food
for its nutrient composition and toxic substances.

(V. P. Gupta)
 PREFACE

Nutrition is the pre requisite for human being which can be achieved by the
consumption of all nutrients viz. carbohydrates, protein, fat, vitamins, minerals and water in
adequate and balanced amount. It is possible only when the foods that we eat have been
impregnated with all nutrients in proportionate amount. Thus the analysis of food is must to
determine the actual nutrient content of the food.

The present manual on Food Analysis has been carefully prepared to determine the
proximate composition of food material in a very easy and simple manner. The procedure
for analyzing some nutrients like soluble carbohydrate, amino acid, vitamin, minerals etc.
have also been mentioned. As well, standardised methods to determine toxic substances
present in the food which sometimes plays a very dangerous role for human health have
been given.

All possible measures have been taken care while preparing this manuals. I hope it
will be useful not only for UG & PG students but for the research worker also engaged in
the field of nutrition and allied field.

(USHA SINGH)

Dept. of Food & Nutrition

College of Home Science
R. A. U. Pusa